Our objective is to understand in precise molecular detail the mechanisms used by DNA polymerases to increase the accuracy of DNA synthesis. At least two mechanisms contribute to fidelity during DNA polymerization, discrimination against errors as the phosphodiester bond is formed (termed base- selection) and 3' exonuclease correction of rare misinsertions or misalignments after bond formation but before further polymerization (proofreading). Both mechanisms are known to operate with a variety of prokaryotic and eukaryotic DNA polymerases involved in the replication, recombination and repair of genetic information. Precise definition of the contributions to fidelity for various mutational pathways by these two mechanisms depends on the ability to probe each one independently at the molecular level. It is now possible to do this using as a model enzyme E. coli DNA polymerase I. This protein has been cloned, sequenced and overproduced (by Catherine M. Joyce at Yale) to provide detailed X-ray cystallographic and NMR structural information on the active sites for both its polymerization activity and its 3'exonucleolytic activity. This information has permitted a single base change to be introduced into the protein coding sequence for the exonuclease active site which inactivates the proofreading activity while not affecting polymerization. We are analyzing this mutant polymerase and contrasting its overall error rate and specificity to that of the wild type protein, using a series of highly sensitive in vitro mutagenesis assays. This approach has allowed us to dissect cleanly, for the first time, the contributions to fidelity of base selection and proofreading. Overall base selection by Pol I achieves an error site of one error for each 10,000 bases polymerized, and proofreading improves fidelity 5- to 10-fold. There are wide variations in these average values depending on the DNA sequence and the type of mutation. This is the first in a series of mechanistic experiments to be performed on the relationship between enzyme active site structures and mutational endpoints.